AIM:
To investigate the inhibitory effect of acetylshikonin on human gastric
carcinoma cell line SGC-7901 and its mechanism.

METHODS:
MTT assay was used to assess the inhibitory effect of acetylshikonin on
proliferation of SGC-7901 cells. Apoptosis-inducing effect was
determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated
dUTP-biotin nick end-labeling with Hoechst staining. Expression of mRNA
and protein in Bcl-2 and Bax was analyzed by reverse
transcription-polymerase chain reaction and Western blot. Antitumor
effect of acetylshikonin on a mouse SGC-7901 model was also determined.

RESULTS:
Forty-eight hours after treatment with acetylshikonin, MTT assay showed
that acetylshikonin inhibited the proliferation of SGC-7901 cells in a
dose-dependent manner. The half maximal inhibitory concentration of
acetylshikonin to SGC-7901 cells was 0.428 ± 0.07 mg/L. Cell shrinkage,
nuclear pyknosis and chromatin condensation, which are the
characteristics of cell apoptosis, were observed in treated SGC-7901
cells and the percentage of apoptosis increased in a dose-dependent
manner. Acetylshikonin down-regulated the expression of Bcl-2 and
up-regulated the expression of Bax in the treated SGC-7901 cells
compared with the controls. The experiment in vivo showed that
0.5, 1, and 2 mg/kg of acetylshikonin significantly inhibited the growth
of tumor in the mouse SGC-7901 model, with an inhibitory rate of
25.00%-55.76%.

CONCLUSION: Acetylshikonin inhibits the growth of SGC-7901 cells
in vitro and in vivo by inducing cell apoptosis.

Although advances have
been made in clinical medicine, no effective treatment modalities are
available for patients with advanced or metastatic tumors[1].
Gastric cancer is one of the most common malignant tumors worldwide, and
its incidence and mortality rank first in China[2,3]. At
present, gastric cancer patients have certain clinical responses to
chemotherapy or radiation therapy, but they cannot tolerate it well[4,5].
Therefore, it is absolutely necessary to explore drugs capable of
preventing and treating gastric cancer and other malignancies.

Chemotherapy is one of the main treatment modalities for advanced cancer[6].
It has been shown that acetylshikonin, one of the shikonin derivatives,
is effective against sterilized and infected wounds[7,8]. It
has been demonstrated that acetylshikonin can inhibit tumor-induced
cutaneous angiogenesis[9] and has obvious antitumor effects
on S180 sarcoma model[10]. However, little is known about the
effect of acetylshikonin on gastric cancer in vitro, especially
in vivo. This study was to evaluate the ability of acetylshikonin
to inhibit cell proliferation and induce apoptosis of human gastric
cancer SGC-7901 cells. Our results demonstrate that acetylshikonin could
induce apoptosis by down-regulating Bcl-2 and up-regulating Bax in
SGC-7901 cells. Significant antitumor effects of acetylshikonin could
also be observed in a SGC-7901 tumor xenograft
model of nude mice.

Effect of
acetylshikonin on the viability of SGC-7901 cells was detected by MTT
assay[11]. Cells were plated in a 96-well plate (5 × 103
cells/well) for 24 h, and then treated with different concentrations of
acetylshikonin (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg/L).
Tetrazolium dye, 3-(4, 5-dimethylthiazol-2)-2, 5-diphenyl-2H-tetrazolium
bromide (MTT, Sigma; 5 mg/mL in PBS) was added to the medium for 4 h,
and measured by spectrophotometry after 15 min at room temperature.
Optical density (OD) which reflects the viable cell population in each
well was determined.

For apoptosis studies,
cells were plated on glass cover slips. Samples were assessed 24 h after
plated for the detection of TUNEL-positivity (brown cellular nuclei
appeared) using an in situ cell death detection kit (Roche
Diagnostic Corp., USA). Apoptotic rates of cells were also determined
with Hoechst staining and cells showing condensed chromatin were defined
as apoptotic[12]. Percentage of apoptotic cells in each
sample was counted under microscope and averaged over 10 fields (80-100
cells each).

Flow cytometry

Cells were harvested and pelleted at 1000 × g for
5 min, then fixed in 70% ethanol and washed with cold PBS. After
suspended in 1 mL propidium, iodide solution (50
mg/mL),
0.1% (w/v) sodium citrate, and 0.1% Triton X-100. Cells were incubated
at 4℃
in the dark for at least 15 min and analyzed with a flow cytometer (FACS,
Beckman Coulter, USA) to determine the number of cells at each cell
cycle and cell apoptosis rate as previously described[11].

Western blot
analysis

Expression of
apoptosis-related proteins (Bcl-2 and Bax) was analyzed by Western
blotting as previously described[11]. In brief, the cells
were lysed in a lysis buffer at 4℃
by sonication. Lysates were centrifuged at 15000
r/min for 15 min at 4℃.
Proteins were separated on SDS-PAGE, transferred to nitrocellulose
filters and incubated with antibodies against Bcl-2, Bax and actin,
respectively. The membrane, were incubated with peroxidase-conjugated
secondary antibodies, and detected with an enhanced chemiluminescence
reagent.

Expression of
Bcl-2 and Bax mRNA

Expression of Bcl-2 and
Bax was semi-quantitatively detected by reverse transcription-polymerase
chain reaction (RT-PCR). Total RNA was isolated from the cells using the
TRIzol reagent (Gibco) and reverse transcribed with the SuperScript
first-strand synthesis system (Invitrogen) according to the
manufacturer’s instructions. Sequences of the primers used for RT-PCR
are listed in Table 1. PCR for Bcl-2 was performed at 94℃
for 3 min, followed by 30 cycles at 94℃
for 30 s, at 70℃
for 30 s, at 72℃
for 45 s and a final extension at 72℃
for 7 min. PCR for Bax was performed at 95℃
for 3 min, followed by 30 cycles at 95℃
for 30 s, at 57℃
for 30 s, at 72℃
for 1 min, and a final extension at 72℃
for 7 min. PCR products were separated on 10 g/L agarose gel stained
with ethidium bromide and observed under ultraviolet light.

In vivo anti-tumor
activity

Seven-week old male
nude mice (supplied by Experimental Animal Center, West China Center of
Medical Sciences, Sichuan University, China) were inoculated
subcutaneously with transplanted human SGC-7901 gastric carcinoma cell
line (5 × 106 cells per mouse). Tumors were allowed to
develop to about 75 mm × 75 mm × 75 mm. Thirty mice were randomized into
6 groups (n = 5). Mice in 3 acetylshikonin treatment groups
received 0.5, 1 and 2 mg/kg acetylshikonin suspended in castor oil; mice
in the castor oil group received an equal volume of castor oil; mice in
control group received an equal volume of normal saline (NS), once a day
for 15 consecutive days. Mice in the positive control group received
cyclophosphamide (60 mg/kg) only on the first day. The animals were
sacrificed and their tumors removed and weighed immediately. Tumor
inhibitory rate was calculated according to the following formula: tumor
inhibitory rate (%) = 1-(Wtreated/Wcontrol) × 100%[11].

Immunohistochemical staining

Thirty tumor tissue samples were fixed in 10% buffered
formalin and embedded in paraffin. Tumor tissue was cut into 4-mm
thick sections. The sections were stained as previously described[13].
Expressions of Bax, Bcl-2 and Caspase 3 were observed under an inverted
phase contrast microscope. Photos were taken and analyzed by Image pro
plus, and the integrated optical density (IOD) was calculated.

Statistical
analysis

Data were expressed as
mean ± SD. Statistical analysis was performed using Student’s t
test and chi-square test. P < 0.05 was considered statistically
significant.

RESULTS

Effect of
acetylshikonin on the growth of SGC-7901 cells in vitro

The SGC-7901 cells were
treated with various concentrations of acetylshikonin for 48 h and cell
viability was determined by MTT assay. Acetylshikonin inhibited the
growth of gastric cancer cells in a dose-dependent manner (Figure 1).
Cell growth was suppressed by 33.7%, 39.3% and 45.5% 48 h after
treatment with 0.2, 0.4 and 0.8 mg/L acetylshikonin, respectively. The
inhibitory rate of 3.2 mg/L acetylshikonin for tumor cell growth was
over 90%. The half maximal inhibitory concentration (IC50) of
acetylshikonin for SGC-7901 cells was 0.428 mg/L.

Acetylshikonin
induced apoptosis of SGC-7901 cells

Twenty-four hours after
exposure to acetylshikonin, SGC-7901 cells began to show morphologic
features of apoptosis and some cells rounded up off the plate,
exhibiting a smaller and circular shape. Cell cycle analysis revealed
that the number of cells was different at the G2/M phase
after exposure to 0.2 and 0.4 mg/L acetylshikonin, but significantly
decreased at the G2/M phase after exposure to 0.8 mg/L
acetylshikonin. Hoechst assay showed nuclear condensation, DNA
fragmentation and perinuclear apoptotic bodies after treatment with 0.2
mg/L acetylshikonin, indicating that acetylshikonin induces apoptosis in
a dose-dependent manner (Table 2).

Acetylshikonin
regulated the expression of Bcl-2 and Bax proteins

Western blot assay
showed that acetylshikonin could induce or inhibit the expression of
Bcl-2 and Bax. Twenty-four hours after treatment with indicated
concentrations of acetylshikonin, the expression of Bax was markedly
increased, while that of Bcl-2 had a trend to decrease. The Bax/Bcl-2
protein ratio was also elevated remarkably. The expression of actin
remained unchanged (Figure 2).

Acetylshikonin
regulated the expression of Bcl-2 and Bax mRNA

The expression of Bcl-2 and Bax was semi-quantitatively
detected with b-actin
as an internal standard. Acetylshikonin inhibited the expression of
Bcl-2 in SGC-7901 cells in a dose-dependent manner. However, the
expression of Bax mRNA increased with the concentration of
acetylshikonin used (Figure 3).

Effect of
acetylshikonin on inoculated tumors in mice

To determine whether
acetylshikonin inhibits tumor growth in vivo, an equal number of
SGC-7901 cells were injected into the right flanks of nude mice. Tumor
growth was notably inhibited in mice after treatment with acetylshikonin
(Figure 4). The inhibitory rate of tumor growth in 3 acetylshikonin
groups and cyclophosphamide group was 55.76%, 39.42%, 25.00% and 58.65%,
respectively. In addition, no toxicity judged by parallel monitoring of
body weight was observed in acetylshikonin-treated mice.
Immunohistochemistry data showed that the IOD value for Bcl-2 was 19.26
± 2.15 in the negative control group and 11.59 ± 1.04 in the 2 mg/kg
acetylshikonin group (Figure 5). However, the expression of Bax and
Caspase 3 was markedly higher in 3 acetylshikonin groups than in
negative control group.

DISCUSSION

Shikonin, isolated from
roots of the medicinal herb Lithospermum erythrorhizon Siebold Zucc[14],
is a Chinese herbal medicine with various biological activities,
including anti-microbial, anti-fungal, anti-HIV, anti-inflammatory,
anti-tumor, and wound-healing activities[15-17]. However, its
toxicity has limited it as a therapeutic agent[14,17].
Acetylshikonin, one of the shikonin derivatives, has been shown to
possess anti-cancer activity[9,10] with less toxicity[18],
which has made acetylshikonin a promising anti-tumor agent. However, its
effects on gastric cancer cells have not been reported.

In
the present study, acetylshikonin suppressed the proliferation of
SGC-7901 cells in vitro, and inhibited tumor growth in a
SGC-7901 tumor xenograft model of nude
mice. Generally, tumorigenesis and tumor progression are strongly
associated with abnormal apoptosis. It has been shown that shikonin and
its derivatives induce apoptosis of tumor cells[16,19,20].
Our data demonstrate that acetylshikonin could also induce apoptosis of
SGC-7901 cells with typical apoptotic alterations, including
morphological changes and positive TUNEL assay, DNA fragmentation and
apoptotic sub-G1 peak.

Apoptosis is a complex process regulated by a variety of factors[21,22].
Members of the Bcl-2 family are the important regulators in the
apoptotic pathway with individual members that promote or suppress
apoptosis. As an anti-apoptotic protein, Bcl-2 confers a negative
control in the pathway of cellular suicide machinery, whereas Bax, a
Bcl-2-homologous protein, promotes cell death by competing with Bcl-2[23,24].
To explore whether apoptosis-related genes contribute to the inhibitory
effect of acetylshikonin on SGC-7901 cells, the relative Bcl-2 and Bax
expression levels induced by acetylshikonin were determined in this
study. We found that acetylshikonin decreased the Bcl-2 expression while
increased the Bax expression in a dose-dependent manner. Moreover,
acetylshikonin up-regulated the expression level of Caspase 3 in vivo.
Taken together, the reduced Bcl-2/Bax ratio, activating Caspases and
cell apoptosis ultimately[24], might serve as a mechanism of
acetylshikonin underlying apoptosis of SGC-7901 cells.

In conclusion,
acetylshikonin has significant antitumor effects both in vitro
and in vivo by inducing apoptosis of SGC-7901 cells involved in
down-regulation of Bcl-2 expression and up-regulation of Bax expression.
Acetylshikonin can be used as a potent and selective agent in the
treatment of human gastric adenocarcinoma.

COMMENTS

Background

Patients with gastric
cancer have certain clinical responses to chemotherapy or radiation
therapy, but they cannot tolerate it. Therefore, it is absolutely
necessary to explore drugs capable of preventing and treating gastric
cancer and other malignancies.

Research frontiers

Previous studies on
shikonin, a chemical derived from a Chinese medicinal herb, have shown
that shikonin has anti-tumor effects although it is toxic.
Acetylshikonin, an acetyl derivative, has less toxicity and suppresses
the growth of sarcomas. However, little is known about its effect on
gastric cancer
in vitro,
especially in
vivo.

Innovations and
breakthroughs

The study demonstrated
that acetylshikonin could exert significant effects on SGC-7901 cells
both in vitro
and in vivo
by inducing cell apoptosis.

Applications

Acetylshikonin can be
used as a potent and selective agent in treatment of human gastric
adenocarcinoma.

Terminology

Apoptosis: A type of cell
death, in which cells use a specialized cellular machinery to kill
themselves, as well as a cell suicide mechanism enabling metazoans to
control the cell number and eliminate them.

Peer review

The study analyzed the
effect of acetylshikonin, derived from a Chinese medicinal herb, on
gastric cancer cell line SGC-7901 in vivo,
which is an interesting finding.